Composition Of Mesophyll Cell Wall Of Ginkgo Leaf And Its Protoplast Isolation Technology

Ginkgo biloba L.,the oldest relict species among existing seed plants,was originated from China and has been widely introduced into many countries and regions around the world because of its special value in medicine,food,decorations,and decoration.Mesophyll protoplasts,as an ideal material of analysing and detecting the structure,composition,and function of cell,provides a new method for the research and diagnosis of the health and aging process of trees and has great application space.But it is difficult to isolate the arbor mesophyll protoplasts because of its specific cell wall structure and composition.At present,there is no research on the isolation of protoplasts from gingko mesophyll cells.Therefore,it has important theoretical significance and application value to study the isolation of protoplasts from gingko mesophyll cells.In the study,we have evaluated different enzyme concentrations,osmotic pressure,p H value,size of nylon mesh,and centrifugal speed on the isolation of protoplast by single control variable based on the study of the cell wall component content of ginkgo leaves.,and the protoplast isolation system for the young leaves of ginkgo was established.Under the conditions of artificial simulated drought-induced senescence,the effects of aging on the isolation of protoplasts from Ginkgo mesophyll and the content of cell wall chemical components were studied.The main results are as follows:1. The results of the mesophyll cell wall components content at different ages（1-year,5-years,10-years,15-years,and 20-years）showed that the content of cellulose,hemicellulose and lignin increased gradually with the increase of leaf age and tree age.In the leaf-expansion period,the cellulose content of the 1-year-old ginkgo leave was the lowest,but the lignin content was the highest,and the cellulose content was about twice that of the lignin content.The content of cellulose and hemicellulose of 5-year-old ginkgo were the lowest.The content of cellulose,hemicellulose and lignin of 10-year-old ginkgo were between 5 and 15 years old.The 15-year-old ginkgo has the highest cellulose content and the 20-year-old ginkgo has the highest lignin content.When the leaf age reached 40 days,the changes of cellulose and lignin content gradually stabilized.In the growing period,the daily increase in cellulose content was the largest（0.2%）,the daily increase in hemicellulose was the lowest（0.02%）.2. The protoplast isolation of 1-year-old ginkgo leaves which growing 2 months showed that the optimal combination of enzyme solution were 2.0%（w/v）cellulase R-10,0.2%（w/v）pectolyase Y-23 and 1.5%（w/v）macerozyme R-10,the optimum concentration of mannitol was 0.7M,The solution p H value was 5.7.After digesting in the dark at 25°C for 5 h,the enzyme solution was purified by filtration and centrifugation,the optimal nylon membrane pore size was 300 mesh,and the centrifugation speed was 50×g.The total protoplast yield reached 5.39×10⁶ g^-1FW,and the protoplast viability reached 80.23%in the first year.In the second year of repeated experiments,the total protoplast yield and viability were 4.98×10⁶g^-1FW and 79.30%.The coefficient of the variation was 4.56%and 0.99%,respectively.After adjusting the concentration of the enzyme solution and increasing the ligninase.The yield and viability of the mesophyll protoplasts at the age of 5 years,10 years,15 years and 20 years of ginkgo leaf expansion period were significantly increased,the protoplast yield and viability of5-year-old ginkgo obtained the highest were 10.16 g^-1 FW and 31.59%,the protoplast yield of15-year-old ginkgo was the lowest(2.17 g^-1FW),and the viability of 20-year-old ginkgo was the lowest（7.8%）.3. The simulated drought experiment of 1-year-old ginkgo proved that ginkgo leaves showed different degrees of senescence with increasing drought-induced senescence levels（0%,10%,15%,20%）and treatment time（5d,10d）,the cell wall component content and protoplast yield and viability of ginkgo leaves also changed greatly.With increasing degree of stress and increasing duration of stress,the content of cellulose,hemicellulose and lignin gradually increased,and the increase of cellulose content was most obvious on the 10th day of stress treatment,up to 3.96%.The yield and quality of protoplasts also gradually decreased with the increase of stress degree and the extension of stress time.